沉默beclin1基因对缺氧缺糖/复氧复糖HT22细胞凋亡的影响

doi:10.16098/j.issn.0529-1356.2020.01.001

摘要/Abstract

摘要：

目的探讨beclin1基因对缺氧缺糖/复氧复糖小鼠海马神经元细胞系HT22细胞凋亡的影响。方法取对数生长期的HT22细胞，随机分为4组：正常组（normal）、缺氧缺糖/复氧复糖（OGD/R）模型组（model）、beclin1基因沉默组（beclin1^-/-）、转染对照组（control）。除normal组外，其余各组细胞均在缺氧缺糖6 h后进行复氧复糖。利用RNAi技术，针对小鼠cDNA序列设计beclin1干扰序列，用脂质体Lipo2000包裹后转染至HT22细胞。于转染48 h后用荧光显微镜观察转染效率，Western blotting检测细胞beclin1 表达情况。各组细胞均于复氧复糖24 h后采用CCK-8法检测细胞活力，乳酸脱氢酶（LDH）法检测细胞损伤情况，免疫荧光染色检测Bax、Bcl-2表达及比值变化，Western blotting检测LC3、P62及Caspase-3表达。SPSS 19.0 统计学软件进行数据分析。结果与normal组相比，model组细胞活力及P62蛋白表达显著降低（P<0.01），乳酸脱氢酶（LDH）漏出率、LC3Ⅱ/LC3Ⅰ、Caspase-3表达及Bax/Bcl-2均显著升高（P<0.01）。与model组相比，beclin1^-/-组细胞活力及LC3Ⅱ/LC3Ⅰ 表达显著降低（P<0.01），LDH漏出率、Bax/Bcl-2及P62、Caspase-3表达显著升高（P<0.01）；control组与model组比较差异无显著性。结论沉默beclin1抑制细胞自噬可使缺氧缺糖/复氧复糖处理的HT22细胞损伤加重，细胞凋亡进一步增加。

关键词: Beclin1, 基因沉, 氧缺糖/复氧复糖, 自噬, 免疫印迹法

Abstract:

Objective To investigate the effect of Beclin1 gene on apoptosis of HT22 of mouse hippocampus neuron treated with oxygen and glucose deprivation/reoxygenation(OGD/R). Methods HT22 cells in logarithmic growth phase were randomly divided into 4 groups: normal, model, beclin1^-/-and control. All groups were reoxygenated after 6 hours of oxygen and glucose deprivation except for normal group. The beclin1 interference sequence was designed for mouse cDNA sequence using RNAi technology, and was transfected into HT22 cells by liposome Lipo2000. The transfection efficiency was observed under fluorescence microscope and Western blotting after 48 hours of transfection. Cell viability was detected by CCK-8 method. Cell damage was detected by lactate dehydrogenase(LDH) method , Bax and Bcl-2 were detected by immunofluorescence staining and the expression of LC3, P62 and Caspase-3 were tested by Western blotting after 24 hours of reoxygenation. SPSS 19.0 statistical software was used for data analysis. Results Compared with the normal group, the cell viability and P62 expression decreased significantly (P<0.01), the LDH leakage rate, LC3Ⅱ/LC3Ⅰ，Caspase-3 expression and Bax/Bcl-2 increased significantly in model group (P<0.01). Compared with the model group, the cell viability and LC3Ⅱ/LC3Ⅰ decreased significantly (P<0.01), the LDH leakage rate, Bax/Bcl-2，P62 and Caspase-3 expression increased significantly in beclin1^-/- group (P<0.01). There was no difference between the control group and the model group. Conclusion Silencing beclin1 inhibites autophagy, which aggravates the damage of OGD/R HT22 cells and further increases apoptosis.

Key words: Beclin1, Gene silencing, Oxygen and glucose deprivation/reoxygenation, Autophagy, Western blotting

中图分类号:

张怡靳晓飞周晓红董贤慧张颖于文涛成媛高维娟. 沉默beclin1基因对缺氧缺糖/复氧复糖HT22细胞凋亡的影响[J]. 解剖学报, 2020, 51(1): 3-8.

ZHANG Yi JIN Xiao-fei ZHOU Xiao-hong DONG Xian-hui ZHANG Ying YU Wen-tao CHENG Yuan GAO Wei-juan. Effect of silencing beclin1 gene on apoptosis of HT22 cells after oxygen and glucose deprivation/reoxygenation[J]. Acta Anatomica Sinica, 2020, 51(1): 3-8.

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